A. Field of the Invention
The present invention relates to methods for detection, differentiation and quantification of T cell populations, comprising the following steps a) contacting a first aliquot of a body fluid of an individual with at least one antigen, wherein the body fluid contains antigen presenting cells (APC) and T cells, b) incubating the first aliquot with the at least one antigen for a definite period of time, c) detection and differentiation of the T cell populations by detecting at least a first marker of the APCs induced by the T cells of a specific T cell population in the first aliquot and in a second aliquot of the body fluid of the individual, which has not been incubated with the at least one antigen, by way of reverse transcription quantitative real time polymerase chain reaction (RT-qPCR), and d) detection and quantification of the T cell populations by determining the ratio of the detected marker of the APCs in the first aliquot to the second aliquot, as well as a kit for performing the method.
B. Related Art
T cells play a key role in the complex network of immune defence against microbial infections and tumour diseases by coordinating the immune response and by controlling and eliminating pathogens as well as tumour cells through manifold direct and indirect effector functions. Interference with the complex T cell function may lead to an erroneous activation of autoaggressive T cells and concomitantly elicit severe autoimmune diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA) and juvenile diabetes (type I diabetes).
In principle T cells exhibit phenotypically a large heterogeneity as well as a broad spectrum of effector functions. Thus, T cells may be roughly classified on basis of the expression of the surface proteins CD4 and CD8 as CD4 positive T cells (T helper cells (Th)) and CD8 positive cytotoxic T cells (CTL).
CD4+ T cells play a key role in the activation, polarisation and coordination of the immune defence. CD4+ T cells are activated due to a specific interaction of their T cell receptor with peptide loaded MHC class II molecules located on the surface of antigen presenting cells (APC) and govern subsequently via cell cell contact and/or secretion of various messenger molecules (for instance cytokines, chemokines) the production of antibodies by B cells (humoral branch of the immune response) and the activation of CTL (cellular branch of the immune response).
CD4+ T cells can be subdivided on basis of the expression of characteristic surface proteins and the production of marker cytokines into T helper 1 (Th-1), T helper 2 (Th-2) and T helper 17 (Th17) cells. Th-1 cells are characterized by the production of the cytokines IFN-γ and TNF-α as well as the expression of the transcription factor T-bet. Th-1 cells support the mounting of an efficient cell-mediated immune response by stimulating the activation and differentiation of macrophages, CD4−CD8+ cytotoxic T cells, CD4+CD8+ cytotoxic T cells as well as of natural killer cells (NK cells) and NKT cells. Th-2 cells are characterized by the secretion of the cytokines IL-4, IL-5, IL-6, IL-10 and IL-13 as well as the production of the transcription factor GATA-3 and support the production as well as the class change of antibodies (humoral branch of the immune system) in B cells. Th-17 cells are characterized by the production of the cytokines IL-17, TNF-α, GM-CSF and IL-6 and seem to play an important role in rheumatologic autoimmune diseases. With regulatory T cells a further CD4+ T cell population aside of Th-1, Th-2 and Th-17 cells was defined, which plays a significant role in the attenuation of immune responses, oral tolerance as well as in the prevention of autoimmune diseases. Regulatory T cells may be subdivided into CD4+ CD25+ CTLA4+ natural regulatory T cells (Treg) as well as into Th-3 and Tr-1 cells, which are characterized by the production of the cytokines TGF-β (Th-3 cells) or IL-10 (Tr-1 cells).
CTL play a central role in combating cells and tissues infected with microorganisms and parasites as well as tumour cells by destroying them via direct effector mechanisms such as the release of cytotoxic substances (e.g. perforin, granzym) and triggering of apoptosis. In addition, by secreting immune stimulatory cytokines (IFN-γ, TNF-α, IL-15) and chemokines (MIP1α, MIP1β, Rantes) as well as various soluble antiviral factors (IFN-α, IFN-β, IFN-δ, CAF) CTLs exhibit further, to some extent very specific effector functions, which contribute very efficiently to restriction of pathogen replication and spreading. Furthermore, additional populations of cytotoxic T cells have been described, which exhibit a CD4+CD8+ phenotype (CD4+CD8dim, CD4dimCD8bright or CD4hiCD8hi).
Thus, T cells represent an important protective mechanism of the acquired immune system for prevention and control of microbial- and in particular of virus-induced diseases as well as for the recognition and destruction of tumour cells.
The activation, polarization and regulation of specific T cells is governed by a strict control via APCs and is essentially defined by the subtype and maturation level of the APCs, by the mechanism of antigen uptake and presentation as well as by the intrinsic properties of the respective immunogen. Hereby the dose and localisation of a immunogen as well as the concentration of the immunomodulatory substances determine, whether a Th-1-, a Th-2- or a Th-17-mediated immune response develops or whether a tolerance is induced.
Professional APCs, such as dendritic cells (DC), monocytes and macrophages, but also B cells take a key position at the juncture between native and acquired immune system by specifically recognizing pathogens and tumour cells, taking these up and presenting fragments thereof together with MHC molecules of class I and class II to T cells. In addition fibroblasts of the skin, epithelial cells of the thymus and the thyroid gland, glial cells, beta cells of the pancreas as well as vascular endothelial cells may act as non-professional APC. Furthermore, current studies show that T cells may also act as APC. These APC T cells are created by the intercellular transfer of MHC class I and class II molecules as well as of costimulatory molecules, such as CD80, CD40 ligand (CD40L), OX40 ligand (OX40L) and 4-1BB ligand (4-1BBL, TNFSF9) due to contact with an APC, in particular a DC (Sokke Umeshappa et al. (2009), J. Virol. 182:193-206).
For a successful stimulation of T cells by APCs three independent signals are required: the specific recognition of peptide loaded MHC molecules via the T cell receptor (TCR) (signal 1), the interaction of APC and T cell based costimulatory molecules with their ligands (signal 2), as well as the presence of T cell polarising cytokines, such as IFN-γ, IL-12, IL-4, IL-6 and TGF-β (signal 3).
Extracellular soluble proteins are usually degraded via the exogenic antigen processing pathway and the resulting peptides are presented complexed with MHC-II molecules on the surface of APCs. Peptides complexed with MHC class II molecules are recognized by CD4+ T cells (T helper cells).
In contrast, degradation of cytosolic proteins occurs via the endogenous processing pathway, leading to presentation of the generated epitopes on MHC class 1 molecules. These peptide/MHC-I complexes are transported to the surface of APCs, where they are presented to cytotoxic T cells (CTL). Although the majority of epitopes presented on MHC class 1 molecules is derived from endogenous proteins and the majority of peptides complexed with MHC-II molecules is derived from exogenous proteins, this distinction is not absolute. For instance, various exogenously existing immunogens, such as particulate structures, various virus particles, immune complexes and lipoproteins end up via a mechanism called cross presentation on the endogenous processing pathway for antigen presentation on MHC I molecules.
For specific activation of naïve T cells is—aside of recognition of MHC molecules loaded with peptides (signal 1)—a second, costimulatory signal required (signal 2). This is triggered by interaction of various APC and T cell based costimulatory ligands with their receptors. Members of the TNF/TNF-receptor super family as well as of the immunoglobulin super family belong to the most important representatives of costimulatory molecules. In absence of the costimulatory signal the T cell becomes anergic. Anergy is the condition in which T cells do not propagate and do not react to an antigen.
The expression of costimulatory signal on APCs is essentially governed via exogenous stimuli, such as components of pathogens or traumatized tissues, as well as by cytokines. The activation and maturation of APCs results in an increased expression of pro-inflammatory and T cell polarising cytokines as well as of costimulatory molecules (CD80, CD86 and CD40), thereby drastically increasing the capability of APCs to activate cell mediated immune reactions. The specific antigen recognition by the TCR and the interaction with costimulatory molecules induces a targeted activation and proliferation of pathogen and disease specific naive T cells. This is accompanied by an increased secretion of IL-2 and the expression of CD40 ligand, which play an important role in the subsequent activation and expansion of other subpopulations of specific T cells. The polarisation of activated T cells in Th-1 or Th-2 effector cells occurs in dependence of the maturation level of the APCs, of the prevalent cytokine milieu as well as of the intrinsic properties and dose of the respective antigen.
The mounting of a specific T cell response in the course of an acute microbial infection usually occurs in three steps: during the effector phase antigen specific naive T cells are activated by contact with APCs loaded with antigen, which leads to a dramatic expansion of the specific T cells, the development of effector functions and the infiltration of activated effector T cells at the site of infection. This effector phase usually extends over a period of 1 to 2 weeks until elimination of the pathogen. In the subsequent contraction phase, which lasts several weeks, over 90% of the produced effector T cells die. Only a few antigen specific T cells survive and differentiate into long lasting memory T cells. In the memory phase these memory T cells persists in relatively stable cell numbers over many years in the body. These memory T cells can quickly be reactivated after a new contact with their antigen and exert their effector functions.
To avoid undesired immune reactions against the bodies own proteins and tissues autoreactive T cells are early on eliminated or inactivated by clonal deletion (anergy). The consequence is an antigen specific tolerance against the bodies own structures, such as proteins, cells, tissues and organs. In autoimmune diseases these protective mechanisms are inhibited or are only insufficiently developed. There is a number of hints that the autoimmune diseases are acquired via an innate “susceptibility”, (genetic disposition), in combination with environmental influences such as microbial infections, pregnancy or due to the similarity of the body's own structures with pathogen and foreign tissue specific polypeptides, the so called molecular mimicry.
Besides, activated T cells play also a central role in the formation of chronic virus infections and the rejection of transplanted tissues and organs.
The determination of the phenotype, the frequency, the specificity, the functionality, the activation status of T cells represents an efficient strategy to gain information about the present course of disease or about diseases already overcome. Furthermore, such methods are of major importance for monitoring (monitoring) specific T cell responses in therapeutic and prophylactic vaccinations, as well as in the diagnostic detection of the number and functionality of T cells in chronic inflammations, autoimmune diseases and in transplant rejection.
In the past decades different technologies for detection of T cells have been developed, which may be roughly divided into two categories. The first group of methods relies on the direct identification and quantification of polypeptides specific T cells by using peptide-MHC multimeres, such as tetramers (Beckman Coulter), pentameres (Proimmune) and streptameres (IBA). This method allows the determination of epitope specific T cells with known MHC restriction. An analysis of the functionality of the T cells is not possible with this method. The most important limitation in using peptide MHC multimeres for the routine monitoring of disease or pathogen specific T cells ensue because the MHC/peptide multimeres are epitope as well as HLA specific. Thus, an exhaustive monitoring of pathogen as well as disease specific T cells in subjects with different HLA constellation requires the use of a broad spectrum of different peptide/MHC multimeres, which brings about high costs. In addition, these peptide/MHC multimeres are so far only available for a limited spectrum of MHC molecules.
A further method for determining specific T cells relies on the use of HLA molecules loaded with peptides, which are linked to green fluorescent protein (GFP). The epitope specific recognition and binding of these complexes by the T cell receptor leads to an internalisation of the GFP labelled peptide, whereby the respective CTL is visualized (Tomaru et al. (2003), Nat. Med. 9:469).
In contrast, the “functional” methods for monitoring specific T cells rely on the ex vivo stimulation of T cell and APC containing patient material with stimulator antigens and the subsequent detection of maturation processes, such as proliferation, production of marker cytokines, in specific reactivated T cells by way of various detection systems. The detection of specific CD4+ T cells is accomplished here usually by stimulation of APC and T cell containing patient samples, such as heparinized whole blood or isolated peripheral mononuclear cells of the blood (PBMC) with proteins, polypeptides or peptides of a length of 15 to 25 amino acids and the detection of the specific T cell activation by determining the production of characteristic marker cytokines or the T cell proliferation. The cytokine detection is done for example with flow cytometry methods, such as intracellular cytokine staining and the cytokine secretion assay as well as with the ELISpot or ELISA technique. The determination of the T cell proliferation may for example be determined by bromodeoxyuridine (BrdU)- or carboxylfluorescein diacetate succinimidyl ester (CSFE) proliferation assays.
In contrast, the detection of specific CD8+ T cells (CTL) is usually done by stimulating APC and T cell containing patient samples with short peptides of a length of 8 to 16 amino acids. Furthermore, CTLs may be detected specifically by infection with recombinant viruses or bacteria, which express the target structures of T cells intracellularly in APCs and the subsequent determination of the marker cytokines produced by the antigen specific T cells, usually IFN-γ by way of flow cytometry methods, such as intracellular cytokine staining or by using the ELISpot or ELISA technology, respectively. In the alternative the specific detection of CTL may be done by means of 51chromium release assays or by using adequate non-radioactive methods, such as the lactate dehydrogenase cytotoxicity assay (for example from Clontech).
These available technologies allow the determination of disease and pathogen specific CD4+ or CD8+ memory T cells, but are not suitable or only very limited suitable for detection of activated T helper cells, which occur only transiently during the active course of disease.
CD4+ T cells are transiently activated during active microbial infections and disease progression. Transient activation implies here that the T cells are only present for a defined, rather short period of time. Thus, activated T helper cells represent an important object for the detection of an active disease incident. A detection assay, which is as significant as possible, is required to determine activated T cells in the context of diagnosis of an active infectious diseases and auto-immune diseases accurately.
However, for certain applications in which discrimination between activated and non-activated T cells is required, such as in the detection of activated T cells during acute microbial infections and reactivations or the detection of activated autoaggressive T cells in the case of suspected multiple sclerosis or type 1 diabetes, are methods which are based on in vitro restimulation of memory T cells not or only very limited employable.
The methods hitherto available for detection of activated CD4+ T cells exhibit so far only a low sensitivity and are thus disadvantageous. The reason for this low sensitivity is inter alia due to the fact that the pathogen or disease specific activated CD4+ T cells are directly detected and are typically present only in small amounts in the patient material. However, this small number of available specific activated T cells hampers reliable and unambiguous detection which also satisfies diagnostic requirements, since the detection limit of these methods available hitherto is frequently undercut.
Thus, the methods available so far for the detection of antigen specific activated T helper cells usually rely for example on the flow cytometry determination of proteins which are transiently expressed on the surface of activated T cells. To these belong in particular the CD40 ligand and the CD25 protein. However, CD40 ligand is hardly detectable by flow cytometry, because the binding of antibodies leads to an internalisation of the CD40 ligand. In contrast, the CD25 protein may not only be found on activated T-helper cells but also on regulatory T cells and is thus not suitable for a reliable distinction between these two subpopulations of T helper cells. In addition, the enhanced expression of HLA-DR and CD69 as well as a reduced expression of CD27 represent further marker for activated T cells. The detection of activated antigen specific T cells requires here a parallel marker detection for determining the specificity, e.g. by using specific tetra-, penta- or streptamers, the phenotype, e.g. by determining characteristic surface markers, and/or the T cell activation, for instance via the production of marker cytokines, after a specific restimulation. A further disadvantage of the methods hitherto known is, that the reliable detection of some marker proteins by way of ELISA, ELISpot or FACS technology is impossible due to the membrane localisation of the marker proteins, the presence of preformed marker proteins in intracellular vesicles, and the high unspecific reactivity of the available antibodies with cellular proteins.
Thus, there exists a need for a method, which allows to detect, to differentiate and to quantify specific T cell populations, which are activated by bacterial, viral, parasitic or autoantigens, thereby enabling the assignment of these T cell populations to specific diseases and disease stages.